This invention pertains to rice plants, plant material and seeds characterized by harboring a specific transformation event particularly by the presence of the bar gene under control of a CaMV 35S promoter, at a specific location in the rice genome. The rice plants of the invention combine glufosinate tolerance with optimal overall agronomic performance, genetic stability and adaptability to different genetic backgrounds.
All documents cited herein are hereby incorporated herein by reference.
The phenotypic expression of a transgene in a plant is determined both by the structure of the gene itself and by its location in the plant genome. At the same time the presence of the transgene at different locations in the genome will influence the overall phenotype of the plant. The agronomically or industrially successful introduction of a commercially interesting trait in a plant by genetic manipulation can be a lengthy procedure dependent on different factors. The actual transformation and regeneration of genetically transformed plants are only the first in a series of selection steps which include extensive genetic characterization, breeding, and evaluation in field trials.
Rice production is commonly threatened by various weeds. Some of these can be highly competitive and in cases of severe infestation can result in yield loss of such magnitude that it makes the crop economically unattractive. For direct-seeded, mechanized rice cultivation typical of temperate production, both cultural practices (e.g. crop rotation, irrigation management) and herbicides are necessary to control weeds (Hill et al. 1994, xe2x80x9cAust. J. Exp. Agric. 34:1021-1029xe2x80x9d).
The bar gene (Thompson et al, 1987, EMBO J. 6:2519-2523; Deblock et al. 1987, EMBO J. 6:2513-2518) is a gene encoding the enzyme phosphinothricin acetyl transferase (PAT), which, when expressed in a plant, confers resistance to the herbicidal compounds phosphinothricin (also called glufosinate) or bialaphos (see also for example U.S. Pat. Nos. 5,646,024 and 5,561,236) and salts and optical isomers thereof. Other genes encoding PAT have been described (see for example: Wohlleben et al., 1988, Gene 70:25-37; EP 275,957; U.S. Pat. No. 5,276,268; U.S. Pat. No. 5,637,489; U.S. Pat. No. 5,273,894).
The transformation of monocotyldenous plants by electroporation of intact tissue capable of forming compact embryogenic callus or compact embryogenic callus obtained from such tissue is described in U.S. Pat. No. 5,641,664. Herein, transformation of compact embryogenic callus of rice by electroporation of a bar gene and the regeneration of transgenic rice plants is disclosed.
Transgenic rice plants containing the gus gene with either the bar gene, or the hyg gene conferring resistance to hygromycin, obtained by the transformation of cells of immature rice embryos by bombardment with DNA-coated gold particles have been described (Christou et al. 1991: Biotechnology 9:957).
Transformation of rice with the bar gene by electroporation of aggregated suspension cells is described in U.S. Pat. No. 5,679,558.
However, the foregoing documents fail to teach or suggest the present invention.
The present invention relates to a transgenic, glufosinate tolerant rice plant, cell, tissue or seed, which is characterized by one or both of the following characteristics:
a) the genomic DNA of the plant, cell, tissue or seed is capable of yielding one or more, such as at least two, advantageously at least three, preferably at least four, for instance at least five, more preferably six of the restriction fragments or pairs of restriction fragments selected from the group of:
i) one EcoRI fragment with a length between about 1159 and about 1700 bp, preferably of about 1327 bp;
ii) one pair of BamHI fragments wherein one has a length between about 805 and about 1093 bp, preferably of about 805 bp and the other has a length between about 1700 and about 2140 bp, preferably of about 2.0 kbp;
iii) one pair of EcoRV fragments wherein one has a length between about 2838 and about 4507 bp, preferably of about 3.8 kbp and the other has a length of more than about 5077 bp, preferably of about 12 kbp;
iv) one HindIII fragment with a length between about 5077 and about 11497 bp, preferably of about 5.3 kbp;
v) one pair of NcoI fragments both with lengths between about 2838 and about 4507 bp, preferably one of about 3.1 kbp and one of about 4.1 kbp;
vi) one NsiI fragment with a length between about 4749 and about 11497 bp, preferably of about 5.1 kbp;
wherein each of the restriction fragments is capable of hybridizing under standard stringency conditions, with the about 1327 bp fragment obtainable by EcoRI digestion of the plasmid having the nucleotide sequence of SEQ ID NO: 1; and/or,
b) the genomic DNA of the plant, cell, tissue or seed can be used to amplify a DNA fragment of between 290 and 350 bp, preferably of about 313 bp, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3, respectively (or includes a DNA fragment of about 290 to about 350 bp, preferably of about 313 bp amplified using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No: 2 and SEQ ID NO: 3, respectively).
The present invention relates to a transgenic, glufosinate tolerant rice plant, cell, tissue or seed, which is characterized in that the genomic DNA of the plant, cell, tissue or seed is capable of yielding at least one, advantageously at least two or more, for instance at least three, preferably at least four, for instance at least five, more preferably six of the restriction fragments or pairs of restriction fragments selected from the group described above comprising the restriction fragments or pairs of restriction fragments described under i), ii), iii), iv), v) and vi) above, whereby the selection can include any combination of i), ii), iii), iv), v) and vi) described above.
The present invention relates to a transgenic, glufosinate tolerant rice plant, cell, tissue or seed which is preferably characterized by both of the characteristics described under a) and b) above.
The invention also relates to the seed deposited at the ATCC under Accession number ATCC 203352, a plant which is grown from this seed, and cells or tissues from a plant grown from this seed. The invention further relates to plants obtainable by propagation of, and/or breeding with a rice plant grown from the seed deposited at the ATCC under Accession number ATCC 203352.
The invention further relates to plants, seeds, cells or tissues (e.g., rice plants, seeds, cells or tissues) comprising herein discussed flanking regions with the 35S-bar gene (as herein discussed) therebetween, or plants, seeds, cells, or tissues (e.g., rice plants, seeds, cells or tissues) comprising a nucleotide sequence which is at least 65%, e.g., at least 75%, such as at least 80%, for instance at least 85%, such as at least 90%, for example at least 95% or even 97% or 100% similar to a sequence disclosed herein, such as the sequences for the flanking regions, or the insertion regions.
The invention further relates to a process for cultivating rice plants of the invention as described above, more particularly a process which comprises applying a herbicide with glufosinate as an active ingredient to the cultivated rice plants.
It is believed that the rice plants of the invention, when cultivated according to the process described above, which comprises applying a herbicide with glufosinate as an active ingredient, display improved growth as compared to untransformed rice of the same cultivar (U.S. Pat. No. 5,739,082). Thus, the invention can comprehend a method for improving the yield or growth of rice plants.
The invention also provides a process for breeding rice which comprises crossing with the rice plants of the invention.
The invention further provides a process for producing a transgenic cell of a rice plant or a plant obtained therefrom, which comprises inserting a recombinant DNA molecule into a part of the chromosomal DNA of a rice cell characterized by the sequence of SEQ ID NO: 4 and, optionally, regenerating a rice plant from the transformed rice cell.
The invention further relates to a method for identifying a transgenic plant, or cells or tissues thereof, which method comprises establishing one or both of the following characteristics of the genomic DNA of the transgenic plant, or its cells or tissues:
a) the genomic DNA of the plant, cell, tissue or seed is capable of yielding at least three, preferably at least four, for instance at least five, more preferably six of the restriction fragments or pairs of restriction fragments selected from the group of:
i) one EcoRI fragment with a length between 1159 and 1700 bp, preferably of about 1327 bp;
ii) one pair of BamHI fragments wherein one has a length between 805 and 1093 bp, preferably of about 805 bp and the other has a length between 1700 and 2140 bp, preferably of about 2.0 kbp;
iii) one pair of EcoRV fragments wherein one has a length between 2838 and 4507 bp, preferably of about 3.8 kbp and the other has a length of more than 5077 bp, preferably of about 12 kbp;
iv) one HindIII fragment with a length between 5077 and 11497 bp, preferably of about 5.3 kbp;
v) one pair of NcoI fragments both with lengths between 2838 and 4507 bp, preferably one of about 3.1 kbp and one of about 4.1 kbp;
vi) one NsiI fragment with a length between 4749 and 11497 bp, preferably of about 5.1 kbp;
wherein each of the restriction fragments is capable of hybridizing under standard stringency conditions, with the 1327 bp fragment obtainable by EcoRI digestion of the plasmid having the nucleotide sequence of SEQ ID NO: 1; and/or,
b) the genomic DNA of the plant, cell, tissue or seed can be used to amplify a DNA fragment of between 290 and 350 bp, preferably of about 313 bp, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID NO: 5 an SEQ ID NO: 2 and NO: 3, respectively.
The invention further relates to a kit for identifying the transgenic plants comprising the elite event of the present invention, said kit comprising PCR probes recognizing the foreign DNA and the 3xe2x80x2 or 5xe2x80x2 flanking sequence of GAT-OS2, preferably having the nucleotide sequence of SEQ ID No. 4 and SEQ ID NO. 5, respectively, for use in the PCR identification protocol.